The process of drug discovery is presently undergoing a fundamental revolution as the era of functional genomics comes of age. The term “functional genomics” applies to an approach utilising bioinformatics tools to ascribe function to protein sequences of interest. Such tools are becoming increasingly necessary as the speed of generation of sequence data is rapidly outpacing the ability of research laboratories to assign functions to these protein sequences.
As bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.
Introduction
Anthrax Toxin Receptor
Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous animals; such as cattle, sheep, horses, mules, and goats. Humans become infected incidentally when brought into contact with diseased animals, which includes their flesh, bones, hides, hair and excrement.
One component of the anthrax toxin has a lethal mode of the action that is not understood at this time. Death is apparently due to oxygen depletion, secondary shock, increased vascular permeability, respiratory failure and cardiac failure. Death from anthrax in humans or animals frequently occurs suddenly and unexpectedly. The level of the lethal toxin in the circulation increases rapidly quite late in the disease, and it closely parallels the concentration of organisms in the blood.
The tripartite toxin Anthrax toxin is produced by Bacillus anthracis, the causative agent of anthrax. It helps the bacterium evade the immune system and can kill the host during a systemic infection. The toxin consists of three proteins: protective antigen (PA) (a single receptor-binding moiety) and two enzymatic moieties, called edema factor (EF) and lethal factor (LF). Once these proteins are released from the bacteria as nontoxic molecules, they move to the surface of mammalian cells and assemble into toxic, cell-bound complexes (Mourez M. et al., Nat. Biotechnol. 2001, 19, 958-961).
Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: edema factor (EF) is an adenylate cyclase that impairs host defenses through a variety of mechanisms including suppressing neutrophil function and impairing host resistance. Lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol (Leppla S H. Anthrax toxin, In Aktories K, Just I, editors. Handbook of experimental pharmacology. Berlin: Springer; 2000, pp. 447-72).
The anthrax toxin receptor (ATR) is encoded by the tumor endothelial marker 8 (TEM8) gene. The anthrax toxin exploits the protein product of the TEM8 gene in order to carry out the first stages of intoxication. The true physiological functions of the ATR is not known, however it has been shown that the TEM8 gene is up regulated in colorectal cancer (St Croix B. et al (2000) Science August 18; 289(5482):1197-1202). TEM8 transcription is also upregulated during angiogenesis. Studies on the mouse tumor endothelial marker 8 (mTEM8) gene, suggests the mTEM8 may be involved in neovascularization (Carson-Walter E. et al. (2001) Cancer Res. September 15; 61(18):6649-55).
The human capillary morphogenesis protein 2 (CMG2) has a similar domain organization to ATR and both sequences share 40% sequence identity over their full length. It has been shown experimentally CMG2 can bind the PA subunits of the anthrax toxin, suggesting CMG2 may be exploited in a similar way to ATR (Scobie H. M. et al (2003) Proc Natl Acad Sci USA. April 29; 100(9):5170-4). The true physiological functions of CMG2 are not known, however it has been shown that a recombinant form of CMG2 is able to bind collagen type IV and laminin (Bell S. et al. (2001) J Cell Sci. August; 114(Pt 15):2755-73).
The anthrax receptor extracellular domain (ANT_IG) is found in the putatively extracellular N-terminal half of the anthrax receptor. Another anthrax receptor domain is found in the intracellular part and is referred to as ANT_C. It is probably part of the Ig superfamily and most closely related to the IPT/TIG domain. The IPT/TIG family consists of a domain that has an immunoglobulin like fold. These domains are found in cell surface receptors such as Met and Ron as well as in intracellular transcription factors where it is involved in DNA binding. The Ron tyrosine kinase receptor shares with the members of its subfamily (Met and Sea) a unique functional feature: the control of cell dissociation, motility, and invasion of extracellular matrices (scattering).
One notable feature of ATR is an extracellular von Willebrand factor A (vWFA) domain, also known as an integrin (I) domain. The vWFA domain is found in many large extracellular proteins. Examples of such proteins include complement proteins factor B (FB), C2, CR3 and CR4, the integrins and collagen types VI, VII, XII XIV (Perkins S J et al, (1994) J Mol Biol. April 22; 238(1):104-19) and anthrax toxin receptors (Bradley K. et al (2003) Biochem Pharmacol. February 1; 65(3):309-14). Functions associated with vWFA domain containing proteins include acting as components of the extracellular matrix, hemostasis, cellular adhesion, and immune defense mechanisms (Colombatti A. et al (1993) Matrix. July; 13(4):297-306).
The vWFA domain found in the integrin class of proteins is referred to as an integrin 1 domain (Roland A et al. (2003) J. Biol. Chem. April 25; 278 (17) 15035-15039). It has been shown that the PA subunit of anthrax toxin binds directly to the ATR vWFA domain in a manner that appears to mimic the binding of integrins to their natural substrate. A soluble form of this domain has been shown to act as an effective extracellular anthrax antitoxin in cell culture (Bradley K. et al (2003) Biochem Pharmacol. February 1; 65(3):309-14).
A motif common to most vWFA domain containing proteins is the Metal Ion-Dependent Adhesion Site (MIDAS). The MIDAS motif is involved in cation (eg. Mg2+, Mn2+, Zn2+ and Ca2+) coordination and is made up of five residues, Asp-x-Ser-x-Ser, Thr and Asp (Scobie H. M. et al (2003) Proc Natl Acad Sci USA. April 29; 100(9):5170-4). A MIDAS motif located within the extracellular vWFA domain of ATR/TEM8 chelates a divalent cation that is critical for PA binding.
It has been shown that the soluble vWFA domain of ATR (sATR) functions to block anthrax intoxication of Chinese Hamster Ovary (CHO) cells in culture (Bradley et al. Nature 2001; 414:225-9; see also WO 04/052277 and WO 02/46228).
Bradley et al. also reviewed the implication of ATR/TEM8/CMG2 in cancer, notably in tumor endothelium, colorectal cancer, bladder cancer, esophageal cancer, lung cancer and melanoma (Bradley et al. Biochemical Pharmacology 2003. Vol. 65. pp. 309-314).
Antisense nucleic acids towards ATR/TEM8 have been suggested to treat anthrax infection as well as cancer (WO 04/013313 and WO 04/052277).
It has been suggested that ATR/TEM8/CMG2 has a natural role in angiogenesis (see Nanda and St. Croix for a review, Current Opinion in Oncology 2004. Vol. 16. pp. 44-49). The extracellular domain of TEM8 was shown to bind to the α3 subunit of collagen VI through the COOH-terminal C5 domain (Nanda et al. Cancer Research 2004. Vol. 64. pp. 817-820). PA might compete with collagen subunits for ATR binding. Nanda et al. suggest that the TEM8/C5 interaction may play an important role in tumor angiogenesis.
Mutations within the CMG2 gene (specifically in the vWFA domain) cause two allelic disorders, juvenile hyaline fibromatosis (JFH) and infantile systemic hyalinosis (ISH; Lacy et al. PNAS 2004. Vol. 101. No. 17. pp. 6367-6372).
In addition, missense mutations within the vWFA domains of various proteins lead to human diseases. Mutation of the Von Willebrand factor precursor (vWF) is associated with the von Willebrand disease (OMIM Acc. No. 193400). Mutation of the Collagen alpha 3 (VI) chain precursor is associated with Bethlem myopathy (OMIM Acc. Nos. 120250 and 158810). Mutation of the Collagen alpha 1 (VII) chain precursor (Long-chain collagen) (LC collagen) is associated with Epidermolysis bullosa dystrophica (dominant, OMIM Acc. No. 120120; recessive, OMIM Acc. No. 131750; pretibial, dominant and recessive OMIM Acc. No. 226600 and OMIM Acc. No. 131850).
Certain proteins that contain one or more copies of the type A domain take part in host defense mechanisms, such as immune response and inflammation (see, for example, Celikel et al., Nature Structural Biology 5: 189 (1998)).
WO 92/17192 discloses a therapeutic composition which is effective in treating or inhibiting thrombosis comprising a monomeric polypeptide patterned on a fragment of wild type mature von Willebrand factor (vWF) subunit. WO 04/062551 relates to polypeptides comprising at least one single domain antibody directed against vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gbIb and/or collagen, homologues and/or functional portions of the polypeptides, for the diagnosis and/or treatment for conditions which require a modulation of platelet-mediated aggregation.
Increasing knowledge of the vWFA domain and ANT_IG domain containing proteins, particularly ATR-like proteins, is therefore of extreme importance in increasing the understanding of the underlying pathways that lead to the disease states and associated disease states mentioned above, and in developing more effective gene and/or drug therapies to treat these disorders.